1. Field of the Invention
The present invention relates to methods for assaying GABA-modulatory compounds for activity as antidepressants, cognitive enhancers, sedative hypnotics, or non-sedating anxiolytics. In particular, the method includes determining efficacy (generally in vitro efficacy) and EC50 values (as used herein incorporating IC50 values) for the compounds at several different cloned (i.e., expressed in cells as directed by heterologous cloned receptor-encoding nucleic acid expression vector molecules) GABAA subtype receptors (each subtype made up of a defined set of specific receptor subunit isotypes). The method optionally includes determining binding affinity of compounds for GABAA receptors. The method results in the development of an activity profile for each compound. As an additional step, animal models predictive of such effects may be used to measure the ability of compounds to effect cognitive enhancement, to act as antidepressants, to mediate sedative hypnotic effects, or to effect anxiolysis in vivo without eliciting certain undesirable side effects.
2. Description of Related Art
Modern drug discovery methodology allows the testing of large numbers of compounds (often assembled into collections termed libraries) for functional characteristics that confer pharmaceutical utility. This xe2x80x9cscreeningxe2x80x9d of such libraries, using specific tests (assays) for functional activity properties, allows the rapid identification of promising compounds for further development as pharmaceutical agents. There has been a longstanding quest in the pharmaceutical industry for new means of identifying such promising compounds. Such new means may involve new assays, or may use old assays to generate data that can be analyzed and applied in new ways to identify compounds with new and useful characteristics.
In the field of psychopharmacology, the use of cloned neuronal receptors as substrates has, provided new, more specific assays with which compounds can be characterized. The use of such receptors has enabled the development of receptor binding profile criteria that are particularly beneficial in the identification of useful psychopharmacological agents. For example, such profiling can identify compounds that will be free of certain undesirable adverse effects (side effects).
The GABAA receptor superfamily represents one of the classes of receptors through which the major inhibitory neurotransmitter, xcex3-aminobutyric acid, or GABA acts. In addition to being the site of neurotransmitter action a number of drugs including the anxiolytic and sedating benzodiazepines bind to this receptor. The GABAA receptor is a chloride channel that opens in response to GABA, allowing chloride to enter the cell. This effects a slowing of neuronal activity through hyperpolarization of the cell membrane potential. GABAA receptors are composed of several protein subunits and are generally pentameric in structure.
A number of cDNAs for GABAA receptor subunits have been cloned. While these subunits share a basic motif of 4 membrane-spanning helices, there is sufficient sequence diversity to classify them into several groups. To date at least 6xcex1, 3xcex2, 3xcex3, 1xcex5, 1xcex4, 2xcfx81, and 1xcfx80 subunit species have been identified; some representing alternatively spliced forms. Native GABAA receptors are typically composed of xcex1, xcex2, and xcex3 subunits, most often in the ratio of two alphas, two betas, and one gamma, although other combinations (some comprising other subunits such as xcex5, xcex4, xcfx81, or xcfx80) have been described. Even if restricted to only xcex1, xcex2, and xcex3 subunits, however, an enormous diversity of GABAA subtype receptors are possible. Evidence such as message distribution, genome localization and biochemical studies suggests that the major naturally occurring receptor combinations are xcex11xcex22xcex32, xcex12xcex23xcex32, xcex13xcex23xcex32, and xcex15xcex23xcex32.
In the typical GABAA receptor, the binding sites for GABA (2 per receptor complex) are formed by amino acids from the xcex1 and xcex2 subunits. Amino acids from the xcex1 and xcex3 subunits contribute to form 1 benzodiazepine site per receptor complex. In a classic allosteric mechanism, the binding of a drug to the benzodiazepine site increases the affinity of GABA binding to the receptor. Benzodiazepines and related drugs that enhance the ability of GABA to open GABAA receptor channels are known as agonists or partial agonists depending on the level of enhancement. Other classes of drugs such as xcex2-carboline derivatives that occupy the same site and negatively modulate the action of GABA are called inverse agonists. A third class of compounds exists that occupies the same site as both the agonists and inverse agonists (blocking access of these agents to the site) and yet has little or no direct effect on GABA activity. These compounds are referred to as antagonists.
The characterization of activities of different subtype receptors has been an area of intense pharmacological discovery. Agonists that act at the benzodiazepine site have long been known to know to exhibit anxiolytic, sedative, and hypnotic effects in animal behavior models, while compounds that act as inverse agonists at this site elicit anxiogenic, cognition enhancing, and proconvulsant effects. While benzodiazepines have long been used as anxiolytics, these compounds exhibit a number of undesirable side effects. These include cognitive impairment, sedation, ataxia, potentiation of ethanol effects, and a tendency for tolerance and drug dependence. Likewise the development of benzodiazepine site ligands for other indications has been thwarted by unfavorable side effect profiles for each indication. For example, compounds known to possess cognition enhancing properties have generally tended to be anxiogenic and proconvulsant, while compounds that produce anxiolytic effects tend to generate unwanted sedation, and do so more powerfully when taken in conjunction with the consumption of alcoholic beverages.
The present invention provides methods for characterizing compounds that act at the GABAA receptor benzodiazepine site. In particular, it provides methods for identifying compounds with characteristics indicating that the identified compounds will exhibit pharmacological properties consistent with their use as antidepressants, cognitive enhancers without anxiogenic or proconvulsant activity, sedative hypnotics without cognition-impairing activity, or non-sedating anxiolytics.
This invention is useful in screening libraries of compounds for therapeutic potential and in drug design efforts.
Measurement of GABA receptor binding affinity is a useful step in any of the methods of the invention. Alternatively, these assays may be performed without measuring the binding affinity of the compound. The assays may include an assessment of the ability of the compound to mediate the desired effects in vivo without eliciting side effects using animal models established to be predictive of the desired effects and animal models predictive of the undesired side effects that have been associated with other compounds acting at GABAA receptors.
As used herein, the term xe2x80x9cefficacyxe2x80x9d refers to amount of potentiation (represented as a % increase, e.g., 10%) or inhibition (represented as a % decrease, e.g., xe2x88x9210%) of GABA activated responses measured for GABAA receptors.
In addition to the ability of a compound to effect a specified magnitude of change in the GABA response at distinct subtype receptors, the EC50 value of the compound at the pertinent receptors is also taken into consideration. As used herein, the term xe2x80x9cEC50xe2x80x9d or xe2x80x9cEC50 valuexe2x80x9d refers to the concentration of a compound needed to elicit half the maximal response (to the agonist or inverse agonist effects of a compound) that can be obtained with the compound. Thus, a compound that exhibits dissimilar EC50 values at different subtype receptors can selectively potentiate one of those receptors over a defined range of drug concentrations, even though the maximal amount of potentiation achievable by the compound is the same for the two subtype receptors over a much broader range of compound concentrations. EC50 values do not necessarily correlate to binding affinities or to compound efficacies.
Cognitive Enhancers: With regard to identifying cognitive enhancers, a method of the invention involves optionally determining the binding affinity of a compound for GABAA receptors having Ro15-1788 binding sites and determining efficacy and EC50 values for the compound at cloned xcex11xcex22xcex32 and xcex15xcex23xcex32 receptors and comparing these values with efficacy and EC50 values for the compound at cloned GABAA receptors containing the xcex12 or xcex13 subunits. The ability of the compound to mediate cognitive enhancing effects may optionally be assessed in vivo by animal model predictive of cognitive enhancement. Whether the compound causes proconvulsant effects may also be assessed in vivo using animal models for detecting proconvulsant activity.
Accordingly, in one aspect this invention provides methods for identifying compounds with cognitive enhancing activity that do not display the side effects of anxiogenesis or proconvulsant activity. These methods comprise:
a) Screening compounds, optionally determining the binding affinity of the compounds for GABAA receptors;
b) determining in vitro efficacy and EC50 values of the compounds using cloned xcex11xcex22xcex32 and xcex15xcex23xcex32 receptors and comparing these values to in vitro efficacy and EC50 values for the compound determined using GABAA receptors that contain the xcex12 or xcex13 subunit; and
c) selecting compounds having significant inverse agonist character and sufficiently low EC50 values at xcex11xcex22xcex32 or xcex15xcex23xcex32 subtype receptors and that produce agonist activity at GABAA subtype receptors that contain the xcex12 or xcex13 subunit.
Thus, the invention presents novel methods for identifying compounds with selective cognitive enhancing properties (1) by examining the binding of a given compound at GABAA receptors and (2) by assessing the ability of the compound to potentiate GABA responses at a series of GABAA subtype receptors. These values are then compared to a set of criteria termed the xe2x80x9cCognitive Enhancer Activity Range Profilerxe2x80x9d (Table I, below). This activity profile comprises measurements of in vitro efficacy (agonist, inverse agonist or antagonist character) and EC50 values at each of 4 GABAA subtype receptors. The activity profile needed for cognitive enhancement is presented as a precise window of inverse agonism at certain subtype receptors and agonism at other subtype receptors. The EC50 criteria at each of these subtype receptors are also presented. Determining efficacy and EC50 values for a test compound is crucial as many compounds bind with high affinity at the benzodiazepine site without potentiating the GABA response at the appropriate subtype receptors.
In certain embodiments, the conclusions drawn from the in vitro determinations of validity may be confirmed by examining the in vivo efficacy of test compounds as cognitive enhancers using animal models for cognitive enhancement.
It may also be necessary to verify that compounds identified by these methods possess the predicted favorable side effect profiles by examining the performance of the compounds in animal models indicative of these side effects. Thus, the animal models may be used as an additional step of the assay to further refine the selection of compounds with cognitive enhancing activity.
Anxiolytics: With regard to identifying anxiolytics, a method of the invention involves optionally measuring the binding affinity of a compound at GABAA receptors having Ro15-1788 binding sites and measuring the efficacy and EC50 values for a compound using cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors and comparing these values with the activities and EC50 values of the compound at cloned GABAA receptors containing the xcex11 or xcex15 subunits. As an additional step, the ability of the compound to mediate anxiolytic effects may be assessed in vivo using an animal model established to be predictive of anxiety, and whether the compound causes sedative effects may also be assessed in vivo by an animal model shown to measure sedation.
Accordingly, this invention provides a method for identifying compounds with anxiolytic activity that do not display the side effects of cognitive impairment, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence or that display these side effects only to a very minimal degree. These method comprise:
a) screening compounds, optionally measuring the binding affinity of the compounds at GABAA receptors;
b) measuring the in vitro efficacy and EC50 values of the compounds at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors and comparing these values to the in vitro efficacy and EC50 values of the compounds at GABAA receptors that contain the xcex11 or xcex15 subunit; and
c) selecting compounds with partial agonist character and sufficiently low EC50 values at xcex12xcex23xcex32 and xcex13xcex23xcex32 subtype receptors that also display lower activity at GABAA subtype receptors that contain the xcex11 or xcex15 subunit.
Alternatively, this assay may be performed without measuring the binding affinity of the compound or with the additional step of assessing the ability of the compound to mediate anxiolytic effects in vivo without causing sedation via an animal model established to be predictive of anxiety and an animal model predictive of sedative effects.
Thus, the invention presents a method for identifying compounds with selective anxiolytic activity (1) by examining the binding of a given compound at GABAA receptors and (2) by assessing the ability of the compound to potentiate GABA responses at a series of GABAA subtype receptors. The resulting values are then compared to a set of empirically defined criteria termed the xe2x80x9cAnxiolytic Activity Range Profilerxe2x80x9d (Table III, below) The criteria given by the Anxiolytic Activity Range Profiler are used to select compounds with anxiolytic activity that have no or very minimal sedative effects. This activity profile comprises determinations of in vitro efficacy (agonist, inverse agonist or antagonist character) and EC50 values at each of 4 GABAA subtype receptors. The activity profile needed for anxiolysis is presented as a precise window of agonism and EC50 criteria at each of these subtype receptors. The combination of determining efficacy, and EC50 values for a test compound is crucial as many compounds bind with high affinity at the benzodiazepine site without potentiating the GABA response at the appropriate subtype receptors.
In certain embodiments, the conclusions drawn from the in vitro determinations may be confirmed by examining the in vivo efficacy of test compounds predicted to have anxiolytic activity in an animal model for anxiety. It may also be desirable to verify that compounds identified by these methods possess the predicted favorable side effect profiles by examining the performance of the compounds in animal models known to be indicative of sedative effects. Thus, the animal models may be used as an additional step of the assay to further refine the selection of compounds with non-sedating anxiolytic properties.
Antidepressants: With regard to identifying antidepressants, a method of the invention involves optionally measuring the binding affinity of a compound at GABAA receptors, measuring the efficacy and EC50 values for a compound at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors, and comparing these values with efficacy and EC50 values for the compound at cloned GABAA receptors containing the xcex11 or xcex15 subunits. Optionally, the ability of the compound to mediate antidepressant effects may be assessed in vivo using one or more animal models predictive of antidepressant activity. Whether a compound produces sedative effects may also be assessed in vivo with an animal model for measuring sedation.
Accordingly in a broad aspect, this invention provides a method for screening compounds for antidepressant activity that do not cause the side effects of cognitive impairment, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence, or that display these side effects only at a very low level. These method comprise:
a) screening compounds, optionally measuring the binding affinity of the compounds at GABAA receptors;
b) determining in vitro efficacy and EC50 values for the compounds at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors and comparing these values to in vitro efficacy and EC50 values for the compounds determined at GABAA receptors that contain an xcex11 or xcex15 subunit; and
c) selecting compounds having partial agonist character and that produce sufficiently low EC50 values at xcex12xcex23xcex32 and xcex13xcex23xcex32 subtype receptors and display lower efficacy activity at GABAA subtype receptors that contain the xcex11 or xcex15 subunit. The compounds selected by this method have antidepressant activity.
The assay may include an assessment of the ability of the compound to mediate antidepressant effects in vivo without causing sedation. Animal models predictive of antidepressant effects and sedative effects can be used for the in vivo determination.
Thus, the invention presents methods for identifying compounds with antidepressant activity, i.e., selective antidepressant activity, (1) by examining the binding of a given compound at GABAA receptors and (2) by assessing the ability of the compound to potentiate GABA responses at a series of GABAA subtype receptors. The resulting values are then compared to a set of criteria termed the xe2x80x9cAntidepressant Activity Range Profilerxe2x80x9d (Table IV, below). The criteria given by the Antidepressant Activity Rang Profiler are used to identify compounds with antidepressant activity that have no or very minimal sedative effects. This activity profile comprises determinations of in vitro efficacy (agonist, inverse agonist or antagonist character) and EC50 values at each of 4 GABAA subtype receptors. The activity profile needed for antidepressant activity is presented as a precise window of agonism and EC50 values at each of these subtype receptors. The combination of determining efficacy and EC50 values for a test compound is crucial as many compounds bind with high affinity at the benzodiazepine site without potentiating the GABA response at the appropriate subtype receptors. Furthermore, as noted previously, EC50 values do not necessarily correlate with binding affinities or compound efficacies.
In certain embodiments, the conclusions drawn from the in vitro determinations may be confirmed by examining the in vivo effects of test compounds selected as having antidepressant activity using animal models for depression. It may also be desirable to verify that compounds identified by these methods do indeed possess the predicted favorable side effect profiles by examining the performance of the compounds in animal models known to be indicative of these side effects. Thus, the animal models may be used as an additional step of the assay to further refine the selection of compounds with selective antidepressant activity.
Hypnotics: With regard to hypnotics, a method of the invention involves optionally measuring the binding affinity of a compound at GABAA receptors having Ro15-1788 binding sites, and measuring the efficacy and EC50 values of a compound at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors and comparing these values with the activities and EC50 values of the compound at cloned GABAA receptors containing the xcex11 or xcex15 subunits. Optionally, the ability of the compound to mediate hypnotic effects is assessed in vivo using an animal model established to be predictive of sedation, and whether the compound causes cognitive impairment may also be assessed in vivo by an animal model shown to be predictive of this effect.
Accordingly, this invention provides a method for identifying compounds with hypnotic activity that do not display the side effects of cognitive impairment, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence or that display these side effects only at a very low level. This method comprises:
a) screening compounds, optionally measuring the binding affinity of the compounds at GABAA receptors;
b) measuring the EC50 and in vitro efficacy values of the compounds at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors and comparing these values to the EC50 and in vitro efficacy values of the compounds that contain the xcex11 or xcex15 subunit; and
c) selecting compounds with sufficiently low EC50 values at xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors, partial agonist activity at xcex12xcex23xcex32 receptors and stronger partial agonist activity at xcex13xcex23xcex32 receptors, that also display lower activity at GABAA subtype receptors that contain the xcex11 or xcex15 subunit as having hypnotic activity.
Alternatively, this assay may be performed without measuring the binding affinity of the compound or with the additional step of assessing the ability of the compound to mediate hypnotic effects in vivo without causing cognitive impairment via an animal model established to be predictive of sedation and an animal model predictive of cognitive impairment.
Thus, the invention provides methods for identifying compounds with hypnotic activity (1) by examining the binding of a given compound at GABAA receptors and (2) by assessing the ability of the compound to potentiate GABA responses at a series of GABAA subtype receptors. The resulting values are then compared to a set of empirically defined criteria termed the xe2x80x9cHypnotic Activity Range Profilerxe2x80x9d (Table II, below). The criteria given by the Hypnotic Activity Range Profiler are used to identify compounds with sedative hypnotic activity that have no or very minimal side effects. This activity profile comprises measurements of in vitro efficacy (agonist, inverse agonist or antagonist character) and EC50 values at each of 4 GABAA subtype receptors. The activity profile needed for hypnotic effects is presented as a precise window of agonism and EC50 criteria at each of these subtype receptors. This well-defined activity profile requires partial agonist activity at subtype receptors containing the xcex12 and xcex13 subunits and lower agonist activity at other subtype receptors. The combination of determining efficacy and EC50 of a test compound is crucial as many compounds bind with high affinity at the benzodiazepine site without potentiating the GABA response at the appropriate subtype receptors.
In certain embodiments, the conclusions drawn from the in vitro determinations may be confirmed by examining the in vivo efficacy of test compounds predicted to have hypnotic properties using animal models for evaluating sedative activity. It may also be desirable to verify that compounds identified by these methods possess the favorable side effect profiles by examining the performance of the compounds in animal models known to be predictive of these side effects. Thus, the animal models may be used as an additional step of the assay to further refine the selection of compounds with selective hypnotic activity.
In a further aspect of the present invention, a method of providing pharmaceutical compounds to patients in need of cognition enhancement, hypnosis, anxiolysis, and/or antidepressant treatment (such patients including humans, pets, livestock, and other animals) is provided. In accordance with this method, compounds are obtained that have been identified as having anxiolytic activity, hypnotic activity, antidepressant activity or cognition enhancing activity in accordance with any of the novel screening, characterization, analysis or identification methods of the present invention. Preferably such screening, characterization, analysis or identification is carried out outside of the United States of America. Once such compounds have been obtained, they are tested, preferably in vivo, for toxicity and pharmacokinetic properties. At least one compound determined to have minimal toxic effects and to have useful pharmacokinetic properties is then selected for clinical development. By useful pharmacokinetic properties is meant pharmacokinetic properties known in the art to be useful for a compound having the particular activity of anxiolytic activity, hypnotic activity, antidepressant activity or cognition enhancing activity, as identified for each particular compound in accordance with any of the novel screening, characterization, analysis or identification methods of the present invention for each compound. By clinical development is meant those activities, including testing in patients, related to the development and submission of information under a United States Federal law which regulates the manufacture, use, or sale of drugs or veterinary products, such as the Federal Food Drug and Cosmetic Act and other applicable government laws and regulations pertaining thereto. The final step in this method is the offer for sale (preferably in the United States of America) for use as a drug or veterinary product of a pharmaceutical preparation (such as a pill, powder, inhalant, elixir, injectible solution, patch or suppository) comprising the compound.
The invention provides methods for screening compounds for activity as antidepressants, cognitive enhancers, sedative hypnotics, or anxiolytics. Therapeutic compounds identified by these methods mediate effects through the benzodiazepine site of the GABAA receptor without eliciting side effects classically associated with compounds exhibiting such activity that act at this site.
Although optional, it is preferred that part b) of each method embodiment (as set forth below) will be conducted on compounds displaying sufficiently potent binding affinities as determined in part a) of the method.
In one aspect of each embodiment of the method, prior to determining an in vitro efficacy value for the test compound, the binding affinity of the compound is measured in cells expressing cloned xcex1, xcex2, and xcex3 GABAA receptor subunits or in a cell membrane preparation of such cells.
In another embodiment, prior to determining the in vitro efficacy values for the test compound, the binding affinity of the test compound is determined in any tissue capable of expressing GABAA receptors containing Ro15-1788 binding sites or in a cell membrane preparation of any tissue capable of expressing GABAA receptors containing Ro15-1788 binding sites. In a preferred embodiment of the method, prior to determining in vitro efficacy values of the test compound, the binding affinity of the test compound is determined in rat spinal cord tissue or in a cell membrane preparation of rat spinal cord tissue.
In more preferred embodiments of the method, the binding affinity of a test compound is measured in rat cortex or in a cell membrane preparation of rat cortex.
In particularly preferred embodiments, the binding affinity of a test compound is determined in rat cortex or in a cell membrane preparation of rat cortex and the test compound is selected for further evaluation if it gives a Ki value of  less than 100 nM or, preferably,  less than 50 nM or, most preferably,  less than 30 nM.
In these embodiments, the binding affinity of a compound may be first determined by evaluating the ability of the compound to displace a radiolabeled compound, for example Ro15-1788 (Flumazenil), known to have high affinity at the benzodiazepine site.
The in vitro efficacy and EC50 value of the test compound may be determined by measuring the chloride flux at the surface of a cell expressing the xcex1,xcex2, and xcex3 subunits of the GABAA receptor in response to GABA by the two electrode voltage-clamp technique. While a variety of cells are suitable for use herein, the preferred cells used in this technique are Xenopus laevis oocytes that have been injected with non-polyadenylated cRNA coding for human derived xcex1, xcex2 and xcex3 GABAA subunits. The preferred form of the xcex3 subunit is the normally expressed long form although an alternatively spliced form may be used.
A: Cognitive Enhancers: Cognitive enhancers identified by this method produce inverse agonist activity at the xcex11xcex22xcex32 or xcex15xcex23xcex32 GABAA subtype receptors and agonist activity at subtype receptors containing the xcex12 or xcex13 subunits of the possible subunit combinations for receptors containing xcex12 or xcex13 subunits, the most relevant are the xcex12xcex23xcex32 and xcex13xcex23xcex32 subtype receptors. Compounds selected according to the invention have EC50 values of about 200 nM or less at the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA subtype receptors and EC50 values preferably of 150 nM or less at these receptors.
The criteria for screening compounds for cognitive enhancing activity are presented below.
Thus, methods of this invention comprise:
a) screening compounds, optionally ones having a binding affinity less than 100 nM or preferably less than 30 nM at any GABAA receptor;
b) determining the in vitro efficacy and EC50 values for the compounds at cloned xcex11xcex22xcex32 and xcex15xcex23xcex32 receptors;
c) determining in vitro efficacy and EC50 values for the compounds at GABAA subtype receptors containing the xcex12 or xcex13 subunit; and
d) selecting a compound having an EC50value determined in b) of less than 200 nM or preferably less than 150 nM, an efficacy value determined in b) of less than xe2x88x925% (e.g., xe2x88x926%, xe2x88x9210%, etc.) or preferably less than xe2x88x9210%, and an efficacy determined in c) of greater than 5% or, preferably, greater than 10%.
In preferred embodiments after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured at cells expressing the xcex11xcex22xcex32 or xcex15xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits  greater than 5% inverse agonist activity ( less than xe2x88x925% efficacy), or preferably  greater than 10% inverse agonist activity ( less than xe2x88x9210% efficacy), at either the xcex11xcex22xcex32 or the xcex15xcex23xcex32 GABAA receptor and gives EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these values are compared to in vitro efficacy values determined using cells expressing GABAA subtype receptors containing xcex12 or xcex13 subunits. Compounds producing  greater than 5% or preferably  greater than 10% agonist activity at these subunits are selected as having cognitive enhancing activity.
In a preferred embodiment, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 value are determined for the test compound using cells expressing the xcex11xcex22xcex32 or xcex15xcex23xcex32 GABAA receptor subunit combinations. If the test compound produces  greater than 5% inverse agonist activity ( less than xe2x88x925% efficacy), or preferably  greater than 10% inverse agonist activity ( less than xe2x88x9210% efficacy), at either the xcex11xcex22xcex32 or xcex15xcex23xcex32 GABAA receptors and gives EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these efficacy values are compared to the in vitro efficacy values determined with cells expressing GABAA subtype receptors containing xcex12xcex23xcex32 or xcex13xcex23xcex32 subunits. Where the compound produces  greater than 5% or preferably  greater than 10% agonist activity at these subunits it is selected as having cognitive enhancing activity.
In alternative embodiments, the method includes an in vivo evaluation of the ability of the compound to mediate cognitive enhancement without causing proconvulsant effects. This is done using animal models predictive of cognition enhancement and of proconvulsant activity. Compounds that produce a statistically significant effect in an animal model predictive of cognitive enhancement are considered to be cognitive enhancing. Compounds that give either a decrease in seizure threshold of less than 25% in the presence of a seizure inducing drug or no significant effect at the p=0.05 level are identified as lacking proconvulsant activity.
In addition, the method may include an evaluation of whether the compound produces anxiogenic effects. This is done using an animal model predictive of anxiogenesis. A compound that gives no statistically significant effect in the animal model predictive of anxiogenesis is identified as lacking anxiogenic activity.
In accordance with another alternative embodiment of the method, the cognitive enhancing properties of the compound are determined without measuring the binding affinity of the compound but with the additional step of measuring the ability of the compound to mediate cognitive enhancement in vivo without proconvulsant effects, via an animal model predictive of cognition enhancement and an animal model predictive of proconvulsant activity.
In accordance with yet another alternative embodiment of the method, the cognitive enhancing properties of the compound are determined without measuring the binding affinity of the compound but with the additional step of measuring the ability of the compound to mediate cognitive enhancement in vivo without proconvulsant effects and without anxiogenic effects, via an animal model predictive of cognition enhancement, an animal model predictive of proconvulsant activity, and an animal model predictive of anxiogenesis, respectively.
The spatial water maze and step-down passive avoidance models are suitable models for in vivo determinations of cognition enhancement. The bicuculline or PTZ seizure threshold tests are suitable for use in vivo to determine proconvulsant activity. The elevated plus maze model is an example of a model that may be used in vivo to predict anxiogenic activity.
B: Hypnotics: Hypnotic compounds identified by this method mediate effects through the benzodiazepine site of the GABAA receptor either without eliciting the side effects classically associated with compounds that act at this site or elicit these side effects only to very low degree. These side effects include cognitive impairment, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence. More specifically, compounds identified as hypnotics by this method show partial agonist activity at xcex12xcex23xcex32 receptors, stronger partial agonist activity at xcex13xcex23xcex32 GABAA subtype receptors, and lower activity at subtype receptors containing the xcex11 or xcex15 subunits. Of the possible subunit combinations for receptors containing xcex11 or xcex15 subunits the most relevant are the xcex11xcex22xcex32 and xcex15xcex23xcex32 subtype receptors. Additionally, compounds useful for any of these indications must have EC50 values of 200 nM or less at the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA subtype receptors and preferably should exhibit EC50 values of 150 nM or less at these receptors.
The criteria for selecting a compound as having hypnotic properties are presented below in tabular form.
Thus in broad aspect, the methods of this invention comprise:
a) screening compounds, optionally, selecting compounds having a binding affinity less than 100 nM at any GABAA receptor;
b) determining in vitro efficacy and EC50 values for the compounds at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors;
c) determining in vitro efficacy and EC50 values for the compounds at GABAA subtype receptors containing the xcex11 or xcex15 subunit;
d) selecting a compound having an EC50 value as determined in b) of less than 200 nM, or preferably less than 150 nM, and an efficacy value for the xcex12xcex23xcex32 receptor of greater than 10%, or preferably greater than 20%, an efficacy value for the xcex13xcex23xcex32 receptor of greater than 50%, or preferably greater than 60%, an efficacy value for the receptor containing the xcex11 subunit of less than 50%, or preferably less than 45%, and an efficacy value for the receptor containing the o subunit of less than 45%, or preferably less than 40%.
In preferred embodiments of the invention, after the binding affinity of the compound has been determined, the in vitro efficacy and EC50 of the test compound are measured using cells expressing the xcex12xcex23xcex32 GABAA receptor subunit combination and cells expressing the xcex13xcex23xcex32 GABAA receptor subunit combination. If the test compound exhibits EC50 values of  less than 200 nM or more preferably  less than 150 nM at these subtype receptors, partial agonist activity at xcex12xcex23xcex32 receptors and stronger partial agonist activity at xcex13xcex23xcex32 receptors, these values are compared to the in vitro efficacy and EC50 values of cells expressing subtype receptors that contain the xcex11 or xcex15 subunits. If the compound also exhibits lower activity at receptors containing xcex11 or xcex15subunits, it is selected as having hypnotic properties.
In other preferred embodiments, after the binding affinity of the compound has been determined, the in vitro efficacy and EC50 values of the test compound are measured using cells expressing the xcex12xcex23xcex32 GABAA receptor subunit combination and cells expressing the xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound gives EC50 values of  less than 200 nM or more preferably  less than 150 nM at these subunit combinations,  greater than 10% or preferably  greater than 20% agonist activity at the xcex12xcex23xcex32 subtype receptor, and 50% or preferably  greater than 60% agonist activity at the xcex13xcex23xcex32 subtype receptor, these values are compared to the in vitro efficacy and EC50 values in cells expressing the GABAA receptors containing xcex11 or xcex15 subunits. If the compound also exhibits  less than 50% or preferably  less than 45% agonist activity at GABAA receptor containing the xcex11 subunit and exhibits  less than 45% or preferably  less than 40% agonist activity at GABAA receptor containing the xcex15 subunit, it is selected as having hypnotic properties.
In more preferred embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values of the test compound are measured using cells expressing the xcex12xcex23xcex32 GABAA receptor subunit combination and cells expressing the xcex13xcex23xcex32 GABAA receptor subunit combination. If the test compound gives EC50 values  less than 200 nM or more preferably  less than 150 nM at these subunit combinations,  greater than 10% or preferably  greater than 20% agonist activity at the xcex12xcex23xcex32 subtype receptor, and  greater than 50% or preferably  greater than 60% agonist activity at the xcex13xcex23xcex32 subtype receptor, these values are compared to the in vitro efficacy and EC50 values at cells expressing xcex11xcex22xcex32 and xcex15xcex23xcex32 subtype receptors. If the compound also exhibits  less than 50% or preferably  less than 45% agonist activity at the xcex11xcex22xcex32 subtype receptor or exhibits  less than 45% or preferably  less than 40% agonist activity at the xcex11xcex22xcex32 subtype receptor GABAA, it is selected as having hypnotic properties.
In alternative embodiments, the method includes measuring the ability of the compound to mediate hypnotic effects in vivo without causing cognitive impairment. This is accomplished using an animal model predictive of a compound""s ability to cause hypnotic effects and an animal model developed to be predictive of cognitive impairment. A compound that shows a statistically significant effect in the animal model predictive of sedation and no statistically significant effect in the animal model predictive of cognitive impairment is identified as having hypnotic properties.
Suitable in vivo animal models include the spontaneous locomotor model for predicting hypnotic effects, and the step-down passive avoidance model or the spatial water maze model determining cognitive impairment.
C: Anxiolytics: Methods are provided for identifying selective anxiolytic compounds. Therapeutic compounds identified by these methods mediate effects through the benzodiazepine site of the GABAA receptor either without eliciting the side effects classically associated with compounds that act at this site or elicit these side effects only to a very low degree. These side effects include cognitive impairment, sedation, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence. More specifically, compounds identified as selective anxiolytics by this method show agonist activity at the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA subtype receptors and lower to no agonist activity at subtype receptors containing the xcex11 or xcex15 subunits. Of the possible subunit combinations for receptors containing xcex11 or xcex15 subunits, the most relevant are the xcex11xcex22xcex32 and xcex15xcex23xcex32 subtype receptors. Additionally, compounds useful for any of these indications must have EC50 values of 200 nM or less at the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA subtype receptors and preferably should exhibit EC50 values of 150 nM or less at these receptors.
The criteria for selecting a compound as having anxiolytic properties are presented below in tabular form.
Thus, in a broad aspect the invention comprises:
a) screening compounds, optionally compounds having a binding affinity less than 100 nM at any GABAA receptor;
b) measuring in vitro efficacy and EC50 values for the compounds at cloned xcex12xcex22xcex32 and xcex13xcex23xcex32 receptors;
c) measuring in vitro efficacy and EC50 values for the compounds at GABAA subtype receptors containing the xcex11 or xcex15 subunit; and
d) selecting a compound having an EC50 as measured in b) of less than 200 nM and an efficacy value as measured in b) is greater than the efficacy values measured in c.
In preferred embodiments, after the binding affinity of the compound has been determined, the in vitro efficacy and EC50 of the test compound are measured at cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits any agonist activity and gives EC50 values  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these values are compared to the in vitro efficacies at cells expressing GABAA subtype receptors containing xcex11 or xcex15 subunits. If the compound exhibits lower or no activity at these latter subunits, it is identified as having anxiolytic properties.
In other preferred embodiments, after the binding affinity of the compound has been determined, the in vitro efficacy and EC50 of the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits any agonist activity and gives EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these values are compared to the in vitro efficacy in cells expressing the xcex11xcex22xcex32 GABAA receptor subunit combination. If the compound exhibits lower to no activity at this subunit it is identified as having non-sedating anxiolytic properties.
In further embodiments, after the binding affinity of the compound has been determined, the in vitro efficacy and EC50 of the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits agonist activity and gives EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, the efficacy values are compared to in vitro efficacy values using cells expressing the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA receptor subunit combinations. If the compound exhibits lower to no activity at these subunits it is identified as having non-sedating anxiolytic properties.
In still other embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured in cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound gives  greater than 30% potentiation (i.e., increase) of the GABA response and EC50 values of  less than 200 nM, or more preferably  less than 150 nM at these subunit combinations, the efficacy values are compared to the in vitro efficacies determined using cells expressing GABAA subtype receptors containing xcex11 or xcex15 subunits. If the compound exhibits lower to no activity (efficacy) at these subunits it is identified as having non-sedating anxiolytic properties.
In yet other embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound produces  greater than 30% potentiation of the GABA response and EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these efficacy values are compared to the in vitro efficacy values determined using cells expressing the xcex11xcex22xcex32 GABAA receptor subunit combination. If the compound exhibits lower to no activity efficacy at this subunit, it is identified as having non-sedating anxiolytic properties.
In more preferred embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound produces  greater than 30% potentiation of the GABA response and EC50 values of  less than 200 nM, or more preferably  less than 150 nM, at these subunit combinations, these values are compared to the in vitro efficacy values measured with cells expressing the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA receptor subunit combinations. If the compound produces lower to no efficacy activity at these subunits, it is identified as having non-sedating anxiolytic properties.
In particularly preferred embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound produces  greater than 30% potentiation of the GABA response and EC50 values of  less than 150 nM at these subunit combinations, the efficacy values are compared to in vitro efficacy measured using cells expressing the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA receptor subunit combinations. If the compound gives  less than 20% potentiation of the GABA response at these latter subunits, it is identified as having non-sedating anxiolytic properties.
In alternative embodiments, the method includes the additional step of measuring the ability of the compound to mediate anxiolytic effects in vivo without causing sedation. This is accomplished using animal models established to be predictive of anxiety and sedative effects. A compound that shows a statistically significant effect in the animal model predictive of anxiety and no statistically significant effect in the animal model predictive of sedative effects is identified as having non-sedating anxiolytic properties.
Suitable in vivo animal models include the elevated plus maze model for predicting anxiolytic activity and the spontaneous locomotor activity model to determine sedative effects.
Antidepressants: A method is provided for identifying antidepressant compounds. Therapeutic compounds identified by this method act through the benzodiazepine site of the GABAA receptor without eliciting the side effects classically associated with compounds that bind at this site. Alternatively, these compounds elicit the side effects only to a very low degree. These side effects include cognitive impairment, sedation, ataxia, potentiation of alcohol effects, and a tendency for tolerance and drug dependence. More specifically, compounds identified as antidepressants by this method show agonist activity at the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA subtype receptors and lower or no agonist activity at subtype receptors containing the xcex11 or xcex15 subunits of the possible subunit combinations for receptors containing xcex11 or xcex15 subunits, preferred for use herein are the xcex11xcex22xcex32 and xcex15xcex23xcex32 subtype receptors. Compounds useful for any of these indications must produce EC50 values of 200 nM or less at the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA subtype receptors and preferably should exhibit EC50values of 150 nM or less at these receptors.
The preferred criteria for screening for compounds having antidepressant activity are presented in the table below.
Thus, the method of this invention comprises:
a) screening compounds, optionally compounds having a binding affinity less than 100 nM at any GABAA receptor;
b) determining in vitro efficacy and EC50 values for the compounds at cloned xcex12xcex23xcex32 and xcex13xcex23xcex32 receptors;
c) determining in vitro efficacy and EC50 values for the compounds at GABAA subtype receptors containing an xcex11 or xcex15 subunit; and
d) selecting a compound having an EC50 as determined in b) of less than 200 nM and an efficacy value as determined in b) greater than the efficacy value measured in c.
In preferred embodiments, after the binding affinity of the compound has been determined, in vitro efficacy and EC50 values for the test compound are measured using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits any agonist activity and produces EC50 values of  less than 200 nM or, more preferably,  less than 150 nM at these subunit combinations these values are compared to in vitro efficacy values determined using cells expressing GABAA subtype receptors containing xcex11 or xcex15 subunits. Compounds giving lower or no efficacy activity at the xcex11 or xcex15 subunits are selected as having antidepressant properties.
In a preferred aspect, the in vitro efficacy and EC50 values are determined for the test compound using xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. If the test compound exhibits agonist activity and EC50 values  less than 200 nM or more preferably  less than 150 nM at these subunit combinations, these values are compared to in vitro efficacy values determined with cells expressing the xcex11xcex22xcex32 GABAA receptor subunit combination. Where the compound exhibits lower or no efficacy activity at the xcex11xcex22xcex32 subunit, it is identified as having antidepressant properties.
In more preferred aspect, the in vitro efficacy and EC50 values determined for the test compound using cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations are compared to in vitro efficacy values determined using cells expressing the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA receptor subunit combinations combination. Where the compound exhibits lower to no efficacy activity at both the xcex11xcex22xcex32 and xcex15xcex23xcex32 subunits, it is identified as having antidepressant properties.
In these embodiments, agonist activity is preferably defined as producing an efficacy value of  greater than 30% potentiation of the GABA response. Thus, in preferred embodiments of the invention if the test compound exhibits  greater than 30% potentiation of the GABA response at the xcex12xcex23xcex32 and xcex13xcex23xcex32 receptor subunits, and EC,5 values of  less than 200 nM or, more preferably  less than 150 nM, at these subunit combinations, these values are compared to the in vitro efficacy at cells expressing the xcex11xcex22xcex32 GABAA receptor subunit combination. Where the compound exhibits lower to no efficacy (agonist) activity at the xcex11xcex22xcex32 subunit, it is identified as having antidepressant properties.
Particularly preferred embodiments comprise: determining the binding affinity of the compound for GABAA receptors determining in vitro efficacy and EC50 values for the test compound with cells expressing the xcex12xcex23xcex32 and xcex13xcex23xcex32 GABAA receptor subunit combinations. Where the test compound produces  greater than 30% potentiation of the GABA response (i.e., agonist activity) and EC50 values of  less than 200 nM or, more preferably  less than 150 nM, at these subunit combinations, these efficacy values are compared to in vitro efficacy values determined using cells expressing the xcex11xcex22xcex32 and xcex15xcex23xcex32 GABAA receptor subunit combinations. Where the compound exhibits  less than 20% potentiation of the GABA response at the xcex11xcex22xcex32 and xcex15xcex23xcex32 subunits, it is identified as having antidepressant properties.
In alternative embodiments, the method includes, an in vivo evaluation of the ability of the compound to mediate antidepressant effects without causing sedation. This is done using animal models predictive of antidepressant activity and sedation. A compound that produces a statistically significant effect in an animal model predictive of antidepressant activity and no statistically significant effect in an animal model predictive of sedative effects is identified as having antidepressant properties.
Suitable in vivo animal models include the Porsolt swim test for predicting antidepressant activity and the spontaneous locomotor activity model for determining sedative effects.
In the forgoing embodiments and in the claims, any of the various criteria presented for characterizing each of hypnotic, antidepressant, anxiolytic or cognition enhancing properties (including those set forth in the various dependent claims submitted herewith regarding a particular one of such properties) may be applied in association with each aspect or embodiment of the invention concerning the characterization of that particular property and to each of the various independent claims submitted herewith regarding the characterization of that particular property.